Malignant transformation represents the phenotypic endpoint of successive genetic lesions that alter gene function and gene regulation in oncogene and tumor suppressor networks. While extensive investigations have characterized protein-coding gene functions in these molecular networks, the functional importance of non-coding RNAs (ncRNAs) is just beginning to be revealed. microRNAs (miRNAs) encode a class of small regulatory ncRNAs with great capacity for post-transcriptional gene regulation by targeting a broad range of mRNAs. Our previous studies identified an important miRNA oncogene, mir17-92, that promotes the development of B-cell lymphomas. mir17-92 encodes a polycistronic miRNA transcript that yields six individual miRNA components, which have distinct biological functions and differential gene regulation during tumorigenesis. This context-dependent differential regulation of polycistronic miRNA components could ultimately determine the oncogenic activity of mir17-92. Aberrant regulation of miRNA biogenesis, stability and function has been frequently observed in human cancer, yet the underlying molecular basis still remains unclear. This is largely due to the lack of an effective methodology to study cancer-related miRNA ribonucleoprotein (miRNP) complexes. Here, we propose to develop a powerful biochemical strategy to isolate and characterize specific miRNA ribonucleoprotein (miRNP) complexes with important functions during tumorigenesis. We engineered a bacterial endonuclease, Csy4, to achieve high affinity binding and inducible cleavage of a specific 16-nucleotide (nt) RNA sequence. Using this 16-nt RNA as a tag, we aim to isolate specific miRNP complexes both in vitro and in vivo. These proposed studies will allow us to identify important miRNP complex components that differentially regulate components of a polycistronic miRNA oncogene in cancer cells. More importantly, our technology can be easily adapted to study cancer related RNP complexes containing other ncRNA species. With the increasing number of functionally important ncRNAs in cancer, our proposed studies will bring fundamental insights into an area of cancer research that has been largely unexplored.